© 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 463-471 doi:10.1242/jeb.093914
RESEARCH ARTICLE
Pharmacological characterization of NMDA-like receptors in the
single-celled organism Paramecium primaurelia
ABSTRACT
Paramecium primaurelia is a unicellular eukaryote that moves in
freshwater by ciliary beating and responds to environmental stimuli by
altering motile behaviour. The movements of the cilia are controlled
by the electrical changes of the cell membrane: when the intraciliary
Ca2+ concentration associated with plasma membrane depolarization
increases, the ciliary beating reverses its direction, and consequently
the swimming direction changes. The ciliary reversal duration is
correlated with the amount of Ca2+ influx. Here, we evaluated the
effects due to the activation or blockade of N-methyl-D-aspartic acid
(NMDA) receptors on swimming behaviour in Paramecium. Paramecia
normally swim forward, drawing almost linear tracks. We observed
that the simultaneous administration of NMDA and glycine induced a
partial ciliary reversal (PaCR) leading to a continuous spiral-like swim.
Furthermore, the duration of continuous ciliary reversal (CCR),
triggered by high external KCl concentrations, was longer in
NMDA+glycine-treated cells. NMDA action required the presence of
Ca2+, as the normal forward swimming was restored when the ion was
omitted from the extracellular milieu. The PaCR and the enhancement
of CCR duration significantly decreased when the antagonists of the
glutamate site D-AP5 or CGS19755, the NMDA channel blocker MK801 or the glycine site antagonist DCKA was added. The action of
NMDA+glycine was also abolished by Zn2+ or ifenprodil, the GluN2A
and the GluN2B NMDA-containing subunit blockers, respectively.
Searches of the Paramecium genome database currently available
indicate that the NMDA-like receptor with ligand-binding characteristics
of an NMDA receptor-like complex, purified from rat brain synaptic
membranes and found in some metazoan genomes, is also present
in Paramecium. These results provide evidence that functional NMDA
receptors similar to those typical of mammalian neuronal cells are
present in the single-celled organism Paramecium and thus suggest
that the glutamatergic NMDA system is a phylogenetically old
behaviour-controlling mechanism.
KEY WORDS: Glutamatergic NMDA system, NMDA receptor
pharmacology, Swimming behaviour, Ciliated protozoa
INTRODUCTION
Glutamate is the most widespread excitatory neurotransmitter
in vertebrates, controlling both peripheral and central
1
Department of Earth, Environment and Life Sciences (DISTAV), University of
Genoa, Corso Europa 26, 16132 Genoa, Italy. 2Department of Pharmacy (DIFAR),
University of Genoa, Viale Cembrano 4, 16148 Genoa, Italy. 3Institute of
Biophysics, CNR Genoa, Via De Marini 6, 16149 Genoa, Italy. 4Institute of Marine
Science (ISMAR), CNR Genoa, Via De Marini 6, 16149 Genoa, Italy. 5Center of
Excellence for Biomedical Research, University of Genoa, Viale Benedetto XV,
16132 Genoa, Italy. 6National Institute of Neuroscience, Corso Raffaello 30, 10125
Torino, Italy.
*Author for correspondence ([email protected])
Received 13 July 2013; Accepted 30 September 2013
neurotransmission. Glutamatergic inputs are mediated by receptors
subdivided into ionotropic and metabotropic receptor families. On
the basis of their pharmacological profile, the ionotropic glutamate
receptors are further subdivided into three subtypes: the α-amino-3hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-preferring
receptor, the kainate-preferring receptor and the N-methyl-Daspartate (NMDA) receptor (Watkins and Collingridge, 1994;
Dingledine et al., 1999). Glutamate and ionotropic glutamatergic
receptors are present in vertebrates as well as in prokaryotes and
plants, suggesting that the glutamatergic system is archaic and
phylogenetically conserved (Tikhonov and Magazanik, 2009).
The NMDA receptor mediates the vast majority of excitatory
neurotransmission. In vertebrates, most of the NMDA receptors are
heteromeric, composed of GluN1 subunits (which bind glycine) in
combination with GluN2 subunits (which bind glutamate). As a
consequence, the activation of these receptors requires the presence
of both glutamate and glycine. The GluN1 subunit arises from one
single gene with eight functional splice variants, whereas GluN2 is
encoded by four different genes (Mori and Mishina, 1995; Zukin and
Bennett, 1995). Recently, a glycine-binding GluN3 subunit, encoded
by two different genes, has been identified. The GluN3 subunit can
assemble with GluN1 to express a functional glycine receptor that is
insensitive to glutamate (Chatterton et al., 2002), but it can also
contribute to the expression of native GluN1/GluN2/GluN3 receptors,
the activation of which results in an NMDA+glycine-evoked current,
which is less sensitive to Mg2+ (Chatterton et al., 2002).
An atypical NMDA receptor-like complex composed of four
proteins – the glutamate-binding protein (GBP), the glycine/N-[1(2-thienyl)cyclohexyl]piperidine(TCP)-binding protein (Gly/TCPBP), the (+)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acidbinding protein (CPP-BP) and the phencycline-binding protein – has
also been described (Ly and Michaelis, 1991; Kumar et al., 1994).
This complex is able to induce the expression of glutamate-activated
ion channels; its activity depends on the presence of glycine and
permits the influx of Ca2+ ions (Aistrup et al., 1996). However, the
exact stoichiometry of subunit assembly is still unknown and it has
not yet been demonstrated whether these subunits can assemble with
GluN subunits to express functional receptors.
The purpose of this study was to verify whether NMDA receptors
are present in the ciliated protozoan Paramecium primaurelia
(Sonneborn, 1975) by evaluating the effects of NMDA receptor
activation on swimming behaviour. The locomotion of paramecia
depends on ciliary movements, which are controlled by the electrical
changes of the cell membrane (Eckert, 1972; Naitoh, 1974). It has
been reported that membrane hyperpolarization is correlated with
augmented ciliary beating and accelerated forward swimming
(Naitoh and Eckert, 1973), whereas depolarization is correlated with
ciliary reversal and backward swimming (Machemer and Eckert,
1973). Paramecia normally swim forward, except for episodic brief
periods of backward swimming or whirling (Jennings, 1906). Upon
463
The Journal of Experimental Biology
Paola Ramoino1,*, Simona Candiani1, Anna Maria Pittaluga2, Cesare Usai3, Lorenzo Gallus1, Sara Ferrando1,
Marco Milanese2, Marco Faimali4 and Giambattista Bonanno2,5,6
RESEARCH ARTICLE
The Journal of Experimental Biology (2014) doi:10.1242/jeb.093914
120
Cells showing PaCR (%)
List of abbreviations
CCR
CGS19755
CPP-BP
continuous ciliary reversal
cis-4-phosphonomethyl-2-piperidine carboxylic acid
(+)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acidbinding protein
D-AP5
D-2-amino-5-phosphonopentanoic acid
DCKA
5,7-dichlorokynurenic acid
EGTA
ethylene glycol tetraacetic acid
GBP
glutamate-binding protein
GluN1
NMDA receptor subunit 1
GluN2
NMDA receptor subunit 2
GluN3
NMDA receptor subunit 3
Gly/TCP-BP glycine/N-[1-(2-thienyl)cyclohexyl]piperidine(TCP)-binding
protein
GRINA
glutamate receptor, ionotropic, N-methyl-D-aspartateassociated protein 1
MK-801
dizocilpine
NMDA
N-methyl-D-aspartate
PaCR
partial ciliary reversal
PCR
periodic ciliary reversal
0
exhibited a PaCR (Fig. 1B–D). Exposure to NMDA concentrations
from 1 to 200 μmol l−1, in the presence of 1 μmol l−1 glycine, produced
a concentration-dependent rise in the percentage of cells that adopt
this spiral-like movement from 13% at 50 μmol l−1 NMDA to 100%
at 200 μmol l−1 NMDA (Fig. 2, Table 1). Fig. 2 also shows that the
exposure of paramecia to increasing concentrations of NMDA in the
absence of exogenously added glycine failed to induce the PaCR in a
significant percentage of cells. In addition, glycine (1 μmol l−1), in the
absence of NMDA, did not lead to any modification of swimming
behaviour in the 80 observed cells (Fig. 2, Table 1).
Ion dependence of the NMDA-induced swimming behaviour
Effects of NMDA receptor antagonists on swimming
behaviour
464
The 200 μmol l−1 NMDA+1 μmol l−1 glycine-induced PaCR was
prevented by 1 μmol l−1 MK-801, a non-competitive NMDA receptor
C
S
S
D
S
Fig. 1. Paramecium swimming behaviour alteration after
NMDA and glycine exposure. Paramecia usually swim
forward (A) but exhibit a spiral-like swimming behaviour due to
a partial ciliary reversal when exposed to 200 μmol l−1
NMDA+1 μmol l−1 glycine in the absence of Mg2+ (B–D). Cells
can immediately start swimming in circles (C), or first swim
forward and then describe circles or loops (B), or, alternatively,
after a lasting ciliary reversal, which terminates as a turn on the
spot, swim forward followed by reversion and a loop movement
(D). The movement was digitally recorded for 60 s at
4 frames s−1. Swimming registration starts at ‘S’.
The Journal of Experimental Biology
In order to analyse the impact of Mg2+ ions on the NMDA-mediated
changes in swimming behaviour, experiments were performed in the
presence of 0.5–3.0 mmol l−1 MgCl2. Regardless of the concentration
used, MgCl2 was unable to trigger ciliary reversal and 2.0 mmol l−1
MgCl2 completely counteracted the NMDA effect (Table 2). The
NMDA+glycine-induced spiral-like Paramecium swimming relies on
the influx of Ca2+ ions. Indeed, paramecia exposed to 200 μmol l−1
NMDA+1 μmol l−1 glycine in medium without Ca2+ and containing
1 mmol l−1 EGTA normally swim forward (Table 2). In contrast, the
omission of Na+ from the extracellular milieu, replaced by an
isosmotic amount of N-methylglucamine, failed to modify the spirallike swimming behaviour induced by 200 μmol l−1 NMDA+1 μmol l−1
glycine (Table 2).
Fig. 1A is a representative image depicting the normal cell swimming
in NMDA-free solution. However, in the presence of 200 μmol l−1
NMDA and 1 μmol l−1 glycine, and in the absence of Mg2+, cells
S
50
100
150
200
NMDA concentration (µmol l–1)
Fig. 2. Dose-dependent effect of NMDA on partial ciliary reversal
(PaCR). Percentage of Paramecium primaurelia cells showing a PaCR when
exposed to NMDA in the presence (open circles) or the absence (filled
circles) of glycine (1 μmol l−1).
RESULTS
Effects of NMDA on swimming behaviour
B
40
0
electrical, chemical or mechanical stimulation, the ciliary Ca2+
channels open, giving rise to a depolarizing Ca2+ action potential
and to a transient increase in intraciliary Ca2+ concentration (Dryl,
1974; Machemer, 1988; Preston and Saimi, 1990). This increase in
internal Ca2+ ions triggers the ciliary reversal. Three forms of
behaviour related to ciliary reversal can be observed: (1) a
continuous ciliary reversal (CCR), yielding relatively long periods
of fast backward swimming mediated by full ciliary reversal, (2) a
periodic ciliary reversal (PCR), characterized by brief and repeated
episodes of backward swimming, and (3) a partial ciliary reversal
(PaCR), giving rise to a spiral-like movement and resulting from the
reversal of only part of the somatic cilia of the cells (Dryl, 1974).
Whereas weak stimuli cause a moderate Ca2+ influx and a short
circular movement, strong stimuli can sustain backward swimming
for tens of seconds or minutes. The duration of backward swimming
is proportional to the duration of the action potential and the addition
of drugs able to reduce the inward calcium current decreases the
duration of stimulated backward swimming (Hennessey and Kung,
1984). Hence, the swimming behaviour of the cell serves as a visual
correlate of the electrical state of the cell membrane.
Our study indicates that the swimming behaviour of paramecia is
modified by NMDA and that these changes are controlled by classic
receptor antagonists, suggesting the existence of NMDA-like
receptors at the cell membrane level.
A
80
RESEARCH ARTICLE
The Journal of Experimental Biology (2014) doi:10.1242/jeb.093914
Glycine
NMDA
(μmol l−1)
0
1
10
50
100
150
200
0 μmol l−1
1 μmol l−1
0% (60)
0% (60)
0% (50)
0% (48)
7% (60)
15% (100)
29% (80)
0% (80)
0% (70)
0% (60)
13% (70)
31% (75)
95% (110)
100% (140)
The number of cells tested in four independent experiments is given in
parentheses.
channel blocker (Fig. 3, Table 3). Fig. 3 and Table 3 also show that
the spiral-like swimming was largely reduced by 40 μmol l−1
CGS19755 and by 200 μmol l−1 D-AP5, two selective antagonists at
the glutamate-binding site located on the GluN2 subunits, as well as
by the selective glycine antagonist DCKA (1 μmol l−1). Prevention of
the NMDA+glycine-induced changes in Paramecium swimming by
the above NMDA receptor antagonists is predictive of the
involvement of GluN2-containing NMDA receptors. Chemical
agents able to discriminate between GluN2A- and GluN2Bcontaining NMDA receptors in mammals are available. In particular,
ifenprodil has been found to preferentially inhibit the mammalian
GluN2B-containing NMDA receptor complex (Williams et al.,
1993), while nanomolar Zn2+ was shown to act as a selective
GluN2A-containing NMDA receptor antagonist (Paoletti et al.,
1997). We investigated the effects of ifenprodil (10 μmol l−1) and
ZnCl2 (100 nmol l−1) on the 200 μmol l−1 NMDA+1 μmol l−1 glycineevoked cell spiral-like behaviour and found that both compounds
were able to counteract NMDA effects (Fig. 3, Table 3). None of the
antagonists administered in the absence of NMDA+glycerine were
effective at the concentration used.
Effects of NMDA on swimming behaviour induced by
membrane depolarization
When P. primaurelia membranes were depolarized using
40 mmol l−1 KCl, a CCR reaction was triggered. The average
duration of the KCl-induced backward swimming amounted to
44.5±1.25 s (N=120 cells). The duration of backward swimming was
dose-dependently increased by NMDA, with an augmentation of up
to 20% by 200 μmol l−1 NMDA+1 μmol l−1 glycine (Fig. 4). Fig. 4
also shows that the CCR duration increment induced by NMDA was
smaller in the absence of glycine. As high NMDA concentrations
could induce receptor desensitization, we then used higher glycine
concentrations (10 and 100 μmol l−1) to prevent NMDA
desensitization. Indeed, it has been found that desensitization
decreases with increasing glycine concentration in cultured
hippocampal neurons as well as in oocytes injected with rat brain
mRNA (Benveniste et al., 1990; Lerma et al., 1990; Mayer et al.,
1989; Nahum-Levy et al., 2001; Vyklický et al., 1990). Our data
indicate that in Paramecium the effects of 1–200 μmol l−1 NMDA
are not modified by increasing glycine concentration from 1 to 10
or 100 μmol l−1 (Table 4).
Co-administration of NMDA and D-serine, a potent agonist at the
glycine-binding site (Mothet et al., 2000), resulted in a significant
increase in CCR duration (Table 5).
As Fig. 5 illustrates, the rise in the duration of the 40 mmol l−1
KCl-induced backward swimming behaviour brought about by
200 μmol l−1 NMDA+1 μmol l−1 glycine was counteracted by the
NMDA receptor antagonists MK-801 (1 μmol l−1), CGS19755
(40 μmol l−1), D-AP5 (200 μmol l−1), DCKA (1 μmol l−1), ifenprodil
(10 μmol l−1) and ZnCl2 (100 nmol l−1).
Identification of NMDA receptor-like complex subunit
encoding genes in the Paramecium genome
We scrutinized the Paramecium genome to see whether it contains
sequences encoding NMDA subunit proteins. The results suggest
that an NMDA-like receptor, with the ligand-binding characteristics
of an NMDA receptor-like complex purified from rat brain synaptic
membranes (Hui et al., 2009) and also found in some metazoan
genomes (see supplementary material Tables S1, S2), is present in
Paramecium. Indeed, searches of the current Paramecium
tetraurelia genome database indicate that Paramecium harbours at
least 20 sequences related to a large family of proteins known as the
transmembrane Bax inhibitor 1 protein family (BI1), some of which
contain seven putative transmembrane helices (Fig. 6;
supplementary material Table S1). In the Paramecium genome we
also identified homologues of glycine-binding protein (GlyBP). The
Paramecium genome carries at least two different sequences in
contrast to the single sequence found in Tetrahymena and in other
organisms (Fig. 7; supplementary material Table S2).
DISCUSSION
Some ciliates have been shown to be sensitive to a variety of
neurotransmitters and neuropeptides, to which they would not
usually be exposed (Le Roith et al., 1980; Nomura et al., 1998).
Treatment of Paramecium with a β-adrenergic antagonist affects its
behavioural responses to depolarization in a time- and dosedependent manner, and the shortening of ciliary reversal duration
occurs concomitantly with impairment of phagocytic activity
(Wyroba, 1989; Ucieklak et al., 1993). In Tetrahymena, the
swimming pattern and the rate of phagocytosis are altered in
response to nanomolar concentrations of opiate endorphins (Renaud
et al., 1991; Renaud et al., 1995). We have previously reported that
GABAB receptors modulate swimming behaviour in Paramecium
by inhibition of dihydropyridine-sensitive calcium channels via Gproteins (Ramoino et al., 2003) and that the number of GABAB
receptors at the plasma membrane is regulated by endocytosis into
clathrin-coated and -uncoated vesicles and by recycling back to the
cell surface (Ramoino et al., 2005; Ramoino et al., 2006).
Glutamate is an important chemo-attractant for paramecia,
probably because it signals the presence of bacteria that are food for
this unicellular organism. This observation therefore implies that
Table 2. Ion dependence of percentage of P. primaurelia cells showing a PaCR when exposed to NMDA
Drugs
−1
−1
200 μmol l NMDA + 1 μmol l glycine
200 μmol l−1 NMDA + 1 μmol l−1 glycine + 2 mmol l−1 MgCl2
200 μmol l−1 NMDA + 1 μmol l−1 glycine + 1 mmol l−1 EGTA + 0 mmol l−1 CaCl2
200 μmol l−1 NMDA + 1 μmol l−1 glycine + 8 mmol l−1 N-methylglucamine + 0 mmol l−1 NaCl2
%Total cells
N
100
0
0
100
140
90
80
60
N, number of cells tested in three independent experiments.
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The Journal of Experimental Biology
Table 1. Percentage of Paramecium primaurelia cells showing a
partial ciliary reversal (PaCR) when exposed to NMDA
RESEARCH ARTICLE
The Journal of Experimental Biology (2014) doi:10.1242/jeb.093914
Increase in CCR duration (%)
NMDA+Gly+ZnCl2
NMDA+Gly+ifenprodil
20
NMDA+Gly+DCKA
40
NMDA+Gly+D-AP5
NMDA+Gly+CGS19755
60
NMDA+Gly+MK-801
80
NMDA+Gly
Cells showing PaCR (%)
100
20
10
0
0
Fig. 3. Effects of NMDA receptor antagonists on PaCR. The 200 μmol l−1
NMDA+1 μmol l−1 glycine (Gly)-induced PaCR was prevented by 1 μmol l−1
MK-801, 40 μmol l−1 CGS19755, 200 μmol l−1 D-AP5, 1 μmol l−1 DCKA,
10 μmol l−1 ifenprodil and 100 nmol l−1 ZnCl2.
0
50
100
Fig. 4. Dose-dependent effect of NMDA on continuous ciliary reversal
(CCR) duration evoked by 40 mmol l−1 KCl. An increase of 13% or 20%
CCR duration was induced when 150 or 200 μmol l−1 NMDA with 1 μmol l−1
glycine was used, respectively. Data are given as means ± s.e.m. (N=60)
from four separate experiments and are normalized to control (40 mmol l−1
KCl) values, taking the control CCR duration as 100 s. Filled circles, NMDA;
open circles, NMDA+glycine.
long-term potentiation and leads to changes in learning and memory
in Aplysia (Ezzeddine and Glanzman, 2003; Murphy and Glanzman,
1997) and Lymnaea (Rosenegger and Lukowiak, 2010). In addition,
an immunocytochemical study reported a specific localization of the
GluN1 subunit in dissociated neurons, the nematocytes and epithelial
cells of the hypostomal region, as well as in the tentacle cells of the
freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa), one of the
first species to develop a nervous system (Scappaticci et al., 2004).
Biochemical and behavioural experiments have provided strong
evidence for the presence of NMDA receptors in hydra tissue and for
their involvement in hydra feeding behaviour (Pierobon et al., 2001;
Pierobon et al., 2004), nematocyst discharge in the tentacles
(Scappaticci and Kass-Simon, 2008) and electrical tentacle activity
(Kay and Kass-Simon, 2009). An NMDA-like receptor has also been
described in the ciliated protozoon T. pyriformis (Fillingham et al.,
2002). It was demonstrated that this NMDA receptor plays a crucial
role in the perception of chemoeffectors (Nam et al., 2007) and that
its stimulation induces an up to threefold increase of intracellular
Ca2+ (Nam et al., 2009). It was found that 1 mmol l−1 NMDA
functions as a repellent, whereas NMDA at 10 μmol l−1 and
10 nmol l−1 functions as a strong attractant, and that 10 μmol l−1
NMDA-induced chemotaxis and intracellular Ca2+ increase are
suppressed by both U73122, an inhibitor of phospholipase C activity,
and wortmannin, an inhibitor of phosphatidylinositol-3-kinase (Nam
et al., 2009).
Table 3. Percentage of P. primaurelia cells showing a PaCR when exposed to NMDA and NMDA receptor inhibitors
−1
−1
200 μmol l NMDA + 1 μmol l glycine
200 μmol l−1 NMDA + 1 μmol l−1 glycine + 1 μmol l−1 MK-801
200 μmol l−1 NMDA + 1 μmol l−1 glycine + 40 μmol l−1 CGS19755
200 μmol l−1 NMDA + 1 μmol l−1 glycine + 200 μmol l−1 D-AP5
200 μmol l−1 NMDA + 1 μmol l−1 glycine + 1 μmol l−1 DCKA
200 μmol l−1 NMDA + 1 μmol l−1 glycine + 10 μmol l−1 ifenprodil
200 μmol l−1 NMDA + 1 μmol l−1 glycine + 10 nmol l−1 ZnCl2
N, number of cells tested in four independent experiments
466
200
%Total cells
N
100
10
20
5
10
10
3
140
70
50
80
60
70
60
The Journal of Experimental Biology
paramecia are endowed with glutamate sensors, possibly receptors.
It is well known that paramecia swim smoothly and quickly in the
presence of glutamate, which indicates a relatively hyperpolarized
membrane potential (Preston and Usherwood, 1988). The
mechanism of hyperpolarization has not been fully elucidated yet
but it probably involves the activation of a hyperpolarizing K+
conductance and shares some interesting characteristics with umami
taste (Van Houten et al., 2000), as a sustained hyperpolarization
induced by glutamate was observed in some rat taste cells (Bigiani
et al., 1997). Glutamate is a broad spectrum agonist that binds all
the glutamate receptors, so that the effects caused by glutamate may
be the result of several events, including the metabotropic receptormediated modification of cell metabolism and the ionotropic
receptor-induced changes of cation balance (Nakanishi et al., 1994;
Traynelis et al., 2010). Therefore, the respective roles that the
different glutamate receptors exert in controlling Paramecium
functions remain a challenging issue. As to NMDA receptors, this
question can be feasibly addressed through the use of selective
NMDA receptor agonists and antagonists.
Evidence is accumulating to suggest the presence and functional
roles of NMDA receptors in invertebrates. They have been
implicated in insect and crustacean neuromuscular transmission
(Ultsch et al., 1993; Feinstein et al., 1998), in insect hormone
production (Chiang et al., 2002), in neuroendocrine function in Ciona
(D’Aniello et al., 2003) and in synaptic activity in the leech (Grey et
al., 2009). Moreover, NMDA receptors mediate learning and memory
in Drosophila (Wu et al., 2007; Xia et al., 2005) and the honeybee,
Apis mellifera (Si et al., 2004), while their role in synaptic plasticity
in Caenorhabditis elegans is still controversial (Kano et al., 2008;
Rose et al., 2005). Disrupting NMDA receptor function prevents
Drugs
150
NMDA concentration (µmol l–1)
RESEARCH ARTICLE
The Journal of Experimental Biology (2014) doi:10.1242/jeb.093914
Table 4. Continuous ciliary reversal (CCR) duration (s) in P. primaurelia cells when exposed to NMDA and glycine
Glycine
NMDA
(μmol l−1)
0 μmol l−1
1 μmol l−1
10 μmol l−1
100 μmol l−1
0
1
10
50
100
150
200
100.00±2.52 (120)
101.21±3.04 (45)
101.80±2.60 (45)
100.62±3.76 (60)
101.04±1.93 (60)
103.38±2.87 (60)
107.24±2.10 (120)
97.69±3.25 (45)
102.70±2.14 (45)
100.40±2.32 (45)
101.02±2.34 (45)
97.45±3.75 (60)
113.51±1.66 (60)
120.08±2.11 (120)
99.69±3.52 (45)
98.98±2.55 (45)
99.65±3.80 (45)
98.98±2.55 (45)
100.70±2.70 (45)
110.89±2.82 (45)
117.38±2.49 (45)
101.21±2.87 (45)
96.42±3.16 (45)
96.39±2.71 (45)
102.21±4.59 (45)
97.77±2.77 (45)
112.64±2.84 (45)
118.14±2.68 (45)
Values (means ± s.e.m.) were normalized to controls (40 mmol l−1 KCl), taking the control CCR duration as 100 s. The number of cells tested in three to five
different experiments is given in parentheses.
NMDA
(μmol l−1)
0 μmol l−1
200 μmol l−1
0
1
10
100
100.00±2.52 (120)
99.69±3.12 (45)
100.05±2.85 (45)
98.75±3.64 (45)
107.24±2.10 (120)
113.88±2.88 (45)
114.94±2.52 (45)
116.44±2.72 (45)
Values (means ± s.e.m.) were normalized to controls (40 mmol l−1 KCl),
taking the control CCR duration as 100 s. The number of cells tested in three
to five different experiments is given in parentheses.
***
***
NMDA+Gly+DCKA
NMDA+Gly+ifenprodil
**
NMDA+Gly+D-AP5
NMDA+Gly+CGS19755
NMDA+Gly+MK-801
NMDA+Gly
10
***
***
***
0
Fig. 5. Effects of NMDA receptor antagonists on CCR. The
NMDA+glycine-induced enhancement of CCR duration evoked by
40 mmol l−1 KCl in P. primaurelia was prevented by exposure to NMDA
receptor antagonists (1 μmol l−1 MK-801, 40 μmol l−1 CGS19755, 200 μmol l−1
−1
−1
−1
D-AP5, 1 μmol l DCKA, 10 μmol l ifenprodil and 100 nmol l ZnCl2). Values
are given as means ± s.e.m. (N=60) from four separate experiments and
represent the percentage change from control (40 mmol l−1 KCl). **P<0.005,
***P<0.0005 compared with control values by Student’s t-test.
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The Journal of Experimental Biology
D-Serine
20
NMDA+Gly+ZnCl2
Table 5. CCR duration (s) in P. primaurelia cells when exposed to
D-serine
containing receptors are to Na+-permeable and Mg2+-insensitive ion
channels (Chatterton et al., 2002). Therefore, our results with Mg2+,
along with the Ca2+ dependency of the effect, indicate the presence of
GluN2 receptor subunits in the Paramecium NMDA-like receptor.
With regard to the GluN2 subunit, the blockade of NMDAinduced changes in swimming behaviour by Zn2+ and ifenprodil
implies the co-presence of the GluN2A and GluN2B subunits
(Neyton and Paoletti, 2006). This condition can be suggestive of a
receptor assembled in a heterotrimeric fashion (GluN1/GluN2A/
GluN2B) although, on the basis of the present results, the presence
of two different receptor populations (GluN1/GluN2A and
GLUN1/GluN2B) cannot be ruled out.
In apparent contrast with our hypothesis, however, searches of the
available Paramecium genome database did not produce sequences
with homology matches to this ionotropic glutamate receptor family,
but revealed the existence of several homologous sequences of the BI1-like protein family as well as of the NMDA receptor-like complex,
the GlyBP. In mammals, the BI-1 family consists of at least six
members: TMBIM1/RECS1, TMBIM2/FAIM2, TMBIM3/GBP (also
known as GRINA, glutamate receptor, ionotropic, N-methyl Daspartate-associated protein 1), TMBIM4/GAAP, TMBIM5/GHITM
and TMBIM6/TEGT (see supplementary material Table S1). GlyBP
is a receptor protein that differs, in terms of length and amino acidic
sequence, from the classic GluN subunits but is able to assemble with
Increase in CCR duration (%)
The major novelty of the present investigation is that Paramecium
is able to sense and respond to the NMDA present in the environment
by modifying its swimming behaviour. Indeed, in the absence of
external Mg2+, NMDA+glycine causes marked functional changes in
Paramecium movements and this action is enhanced by membrane
depolarization. Addition of K+ at a high concentration to the medium
elicits membrane depolarization, thus causing backward swimming
through the opening of voltage-dependent Ca2+ channels in the ciliary
membrane (Oami and Takahashi, 2002). NMDA affects the
behavioural response to membrane depolarization by increasing
backward swimming duration. These changes were relieved by the
selective NMDA receptor channel blocker MK801, indicative of the
involvement of NMDA receptors in the observed effects. Notably, the
modification of Paramecium swimming was also counteracted by DAP5 or by DCKA, selective antagonists of the glutamate-binding sites
located at the GluN2 subunits and of the glycine-binding sites at the
GluN1 subunits, respectively. Our findings imply that the co-presence
of the two agonists is required for NMDA-induced reversal of
swimming direction. Hence, NMDA alone (i.e. in the absence of
glycine) or glycine alone (i.e. in the absence of NMDA) produced
little or no change in Paramecium movement. D-Serine, a glialderived endogenous ligand for the glycine-binding site of NMDA
receptors in mammals (Mothet et al., 2000; Oliet and Mothet, 2009;
Schell et al., 1997), behaved similar to glycine thus strengthening the
role of this binding site in the machinery of the NMDA-like receptors
in Paramecium.
The above-mentioned NMDA-induced changes were observed in
the absence of external Mg2+, an ionic condition widely used to force
NMDA receptor activation. Accordingly, in our experiments the
introduction of Mg2+ abolished the effects of NMDA. NMDA
receptors are channels permeable to both Na+ and Ca2+ (Ascher and
Nowak, 1986; MacDermott et al., 1986; Mayer and Westbrook, 1987),
and usually blocked by Mg2+ (Ault et al., 1980; Nowak et al., 1984).
In particular, GluN2-containing receptors are Ca2+ permeant and
highly sensitive to Mg2+ (Paoletti and Neyton, 2007), while GluN3-
RESEARCH ARTICLE
The Journal of Experimental Biology (2014) doi:10.1242/jeb.093914
TM1
TM2
TM3
TM4
Pt1
Pt2
Pt3
Pt4
Pt5
Pt6
Pt7
Pt8
Pt9
Pt10
Pt11
Pt12
Pt13
Pt14
Pt15
Pt16
Pt17
TETH1
TETH2
TETH3
TETH4
TETH5
TETH6
TETH7
TETH8
TETH9
TETH10
HsTMBIM1
HsTMBIM2
HsTMBIM3
HsTMBIM4
HsTMBIM5
HsTMBIM6
RNLFSKLFLQMIIICVY-----------------------VWIVHSIPAL--DHF--LEET-----KW------IFWLSLGICIGTATLALIYRNRITVSPTNWLVFIVF-----------TLSFASVCGCLVAFG-----------NSQIGLLLFVNFA
QLLFVFVDNRLGIY--------------------------VWIVHALPDL--DHF--LENT-----KW------IFWLSLGICIGISILAFIYRNHIKTTPTNWLVFLLF-----------TLSLASVCGCLVAFG-----------NSQVGLLIFVNFA
RKLYQYLLIQMIVISLF-----------------------IYWVYASPSL--DSY--LEGK-----PW------LFWLCLFISIGTATLALIFRKDVAVF PWNWVVFVTF-----------TLSVSVVCGTLVALG-----------DSVIGLLVFSSLA
VKVFVLLA--LMLTTFY-----------------------VMAFIFLVKK--VQI--IKNG-----EIVQYHFIIYILSCVITILMGRWAY-FSESS RKFPLNIFCYLFF-----------TSGAAYVFGQPLSLI--LQGGYYSGQDWIILLYLSTMTL
RKVFLIILFQIGFTFIT-----------------------TLIAYSQIPI--IDS--LCSR-----PL------LFWIFIVVLILVIFL LMRFQKLAKQHPYNYICYSSF-----------TLSISYLFFYTIHHYPT---------YSNHIISLITLQF
LKVYIFLTMELAFTFLLVILGLYTNMQQWLVTTGQEESCYCAFGSFSQCG--CTYISHYDS-----TW------LFWVSIVFSLILHLI LFCGNQRVRQKPWNFIILGLY-----------ILFFGFLVTNLCIIIAYEF-------GVGIVWQAIGITF
KKVYTLLTMELLITLGMIALGLYTGMANWLVQIELDDDVILCYYQAFGPMYCESY--HYQTIQPYPTW------LFYVSFFVALIMQCA LYCGGNLARKAPVNYIVLFLY-----------IVFFGFTLTTFCILMAMYW-------GQAIVWQAWGITF
KKVFSIVGFQLLATSAVAYS--------------------AMNYNFIAEL--CEY-------------------LYIPAIIGSIVTGLWIYLSPSSA RRFPKNYILLSVF-----------TLSEAIALAITCSAI----------GDPEIIFQAFIITT
RKVYSIMILQLILTVVA-----------------------CCFSYFCIPY--RDF--QNDH-----SG------WVYLAIAIAIIIELILLWIPKYSWRVPHNYLFLFVF-----------TLAESYVISQLCSYVFNK--------YRFIVLMAAALTL
RKVYSIMILQLLLTVAA-----------------------CCLSYFWIPY--RDF--QNEY-----SG------WVYLAIAVAIIIEIILLWIPKYSWRVPHNYLFVFVF-----------TLAESYTISQLCSYVFNK--------YRFIVLMAAALTL
RKVYAIMIIQLFITMIM-----------------------CLNSYLSLDY--RRF--QLQN-----TG------YAYLALAISIFVELLLFCIPKFAWRVPYNFILLFIF-----------TLCEGYLISSLCSYVFDKYSE----NGGFIVLMAASLSL
KKVYSIMIIQLFLTMIM-----------------------CLISYLSLDY--RRF--QLDH-----SG------FAYLALGISIFIELLLFCVPKFAWRVPYNYILLFIF-----------TLCEGYLISNLCSYVFDRYSD----NGGFIVLMAASLSL
KKVYSIMIIQLLITMIM-----------------------CVISYVSIDY--RMF--QLQH-----SG------YAYLALGIAIFIEVILFCIPKFAWRVPYNYLLLLIF-----------TVCEGYLISNLCSYVFDEYSQ----NGGYIVLMAASLSL
RKVYSILSLQLLFTALL-----------------------TIWCITQEPV--KNF--VVQQ-----II------LFVLAAITAIVLMCVLLCCKANARKAPKNYILLSLF-----------TFCEAYVVAFICCSTATENS-----NGIEIIVIALSMTV
VKVYAIMSFQLSITFLL-----------------------ILASYYFQNV--RNA--IINTSTIQYTP------LTIFCFVIALVIEVAIF CCRKVARKVPLNYILLTIF-----------TLCFSTVVAAPCIICFELLS-----NGVQLVIIAASITV
VKVYSLLTIQLFVTFVM-----------------------VAIACFSKAF--RDL--LINPYSYKATP------FYWSMFAVSFVTEIAIF CFKKVARKVPNNYIALTIF-----------TVSFSFVVAGSCAVCKDAFEN-----GGTLILIAALMTF
RKVYLILSFQLLFTTIF-----------------------CTFSYFSTGF--AVY--QLQN-----TW------LFYVLLIVGLICEIS LICCKNVSRKVPNNYIILGVF-----------TFCESWIVSYSCSIAYLIYPE----NGGQLVLIAAVLTL
RKLYFALFMQLLIIGIF-----------------------AFFVHLYENL--RVH--LQQN-----YV------YFWVSLAGAAIIGILGLFFRQAI RKSPINYICMFLW-----------TIFFGTILVYAVSIN----------KDSIVGLMVFVLLA
RKVLGIICAQLIITTLF-----------------------ILVGVFSPTY--QNF--QQNN-----KW------LTIFCLLLNIALLFALYCFRDFCRQVPKNYILLFLY-----------TFSESFLISYLCGVT-----------NPTVVLLAGALTT
KKVFCIVFFQLLVTSIF-----------------------SALSMYVIHF--QSF--QVEY-----YA------LLFVALGLIIITQISVFASRNAARKVPLNYILLLLF-----------TISWAYLVSFLCGGFSINQDGTYNERNQTIIFLSVIVTF
RKVLGIICTQLIITFAF-----------------------IIPSTLSQDY--RDF--QKRY-----IF------IAYLSLILNIATMITLYCFRKQCMKVPNNYILLFIF-----------TITEGYLISMITSVS-----------QPEVVLLAGGITF
RK--------LAFTALM-----------------------IFMSQQSRSF--RLF--QAQN-----VW------LFTLSTVLTVAISIGMYCVPALTKKVPINYFALGLF-----------TVCEGYTVSAFTLQY-----------SKLVVLQAGFLTA
RKVYLILGTQLLVTVLM-----------------------TVGAMYSPGF--TTF--QQNN-----LW------LLYTCIVIMFIVEIALFCFRNIAKTVPINYICLFIF-----------TFCMSYFVSTCCSLLNKSSED-----GQKMILVAAVMTF
RKVYLILGAQLLVTVLM-----------------------TVGAMYSPGF--TTF--QQNN-----LW------LLYTCIVVMFIVEIAILCFRNVARTVPTNYICLFIF-----------TFCMSYLVSACCSVVKQQSDD-----GQKTVLIAAVMTL
TKVYSILSVQIGITCAM-----------------------CFIAIENSGF--NSFLKDSSN-----LW------LFYVSIVMTLILCIMIVCYRKFAREVPTNYICLFLF-----------TLFESYIVAQICVLY-----------SPRIVIMAALLTM
TKVYTILSAQMAVTVIL-----------------------CAYSMSSQKF--KNF--QLNN-----PG------LMIAALVVNIICLLVLICSRDQARKVPNNYILLGVF-----------TLCESYLVSFICSMS-----------NPKIVFLAALFTM
RKVYSILLTQLLLTALV-----------------------CYAGMYNPTF--GAY--LITS-----PA------TLVLSIIVSLSILLAMF CNKNVSRIVPANYILLGLF-----------TVCESYIVSFFCALISWTESGQPDYEGRNLVLLAAFFTI
RKVYSIISVQLLITVAI-----------------------IAIFTFVEPV--SAF--VRRN-----VA------VYYVSYAVFVVTYLILACCQGPRRRFPWNIILLTLF-----------TFAMGFMTGTISSMY-----------QTKAVIIAMIITA
RKVYTILLIQLLVTLAV-----------------------VALFTFCDPV--KDY--VQAN-----PG------WYWASYAVFFATYLT LACCSGPRRHFPWNLILLTVF-----------TLSMAYLTGMLSSYY-----------NTTSVLLCLGITA
RKVFLVLTLQLSVTLST-----------------------VSVFTFVAEV--KGF--VREN-----VW------TYYVSYAVFFISLIVLSCCGDFRRKHPWNLVALSVL-----------TASLSYMVGMIASFY-----------NTEAVIMAVGITT
RKVYSILSLQVLLTTVT-----------------------STVFLYFESV--RTF--VHES-----PA------LILLFALGSLGLIFALILNRHKY---PLNLYLLFGF-----------TLLEALTVAVVVTFY-----------DVYIILQAFILTT
HSTYMYLAGSIGLTALS-----------------------AIAISRTPVL--MNF--MMRG-----SWV-----TIGVTFAAMVGAGMLVRSIPYDQSPGPKHLAWLLH------------SGVMGAVVAPLTIL------------GGPLLIRAAWYTA
KKVYASFALCMFV---------------------------AAAGAYVHMV--THF--IQAG---------------LLSALGSLILMIWLMATPHSHETEQKRLGLLAGF-----------AFLTGVGLGPALEFCIAV--------NPSILPTAFMGTA
Pt1
Pt2
Pt3
Pt4
Pt5
Pt6
Pt7
Pt8
Pt9
Pt10
Pt11
Pt12
Pt13
Pt14
Pt15
Pt16
Pt17
TETH1
TETH2
TETH3
TETH4
TETH5
TETH6
TETH7
TETH8
TETH9
TETH10
HsTMBIM1
HsTMBIM2
HsTMBIM3
HsTMBIM4
HsTMBIM5
HsTMBIM6
SLIFFLFLYS-S--TVRRKITYSGAVLFVSASILIVFELF----TI--FT-KISL----FWIMFISLSS-FLFAFLLLYDTYTNLNCGDSYDINKA------DDVSGSVTIYWDII----LLFLKMAELIKD
SLIFFLFLYS-S--TVRRKITYSGAVLFVSAGILIVFEMF----TI--FS-QISL----FWIMVISLSS-FLFAFMLLYDTYSNLNSEDSYDINTA------DDVSGSVTIYWDIV----LLFLKMTELIKD
SMVFFLFMYS-L--TVKRRLTYQGSILFISASILLIFEIF----TI--FT-EVSL----FWLSSVSLFA-FLLAFLLIYDTYTNVNSGDQYDVNQA------DEVSGSVIIYWDVI----LLFLKMNELIKD
GTYLCIIIFTFCRQTPQKPYIIIFSVVGVMIFLLFIFLLT----AP--YY---------LGLLVASFACHVLYGCLLVIDIKLIT--QGKFSLRTN------QYVSGALYLYLDIT----FMIFYFIGCILA
GIIISLLAYS-Y--FTASEINLNKGLTFILITIALLFIFL----FL--YF-ELSL----KFLFILSFLI-ILYGVHIIIDTLLIV-NGEKHELDID------DYIIAALMTQVDIIGLISILFQKLLSQISK
GFVLALTAYS-F--KTKTSFTFGIGSIFLLTPTLVLMLIL----MG--VY-SQFA----LSIFLCTLLV-VGQGFFLIWETKAII-GDGKLKLSID------DYVIGSLLLYGSII----QLLWRIMMLIIA
IIVLALTLYA-C--KTKTDFSFKIGAIFILCPTILMLAIM----LC--IW-WSYA----VYILLCTLFI-VIYGFYLIWETQLIM-GKGKLKLSID------DYVIGSLLLYATII----QLFLRIIEILAI
GIVISLATYA-M--TTKNDLSYHGAAIFLLSFGCLMAGLT----YF--IF-RSSF----AYQIYLIGGA-ISLGFYLVYDIQLII-GDKQLRLTVD------DYVLGSIMIYTDII----KIFIRVVKILMK
AAVIGLTLYA-C--KTKKDFTTKGAFLFMASTSLFLFAIL----SG--VY-YDQA----MSLLYSLISS-LLFGVYLIYDTQLII-GGSTHKLSID------DYIIGAMFIYIDIV----YLFAHIVLIIVA
AAVIGLTLYA-C--KTKKDFTTKGAFLFMASTSLFLFAIL----SG--VY-YDQA----MSLLYSLISS-MLFGIYLIYDTQLII-GGSTHKLSID------DYIIGAMFIYIDII----YLFAHIVLIIIA
AAVIGLTLYT-C--KTKSDYTTKGALLFMCVTSLLLFGIM----AG--VY-YQNV----INLIYSLLCC-LLFGAYLIYDTQLIL-GGSTHKLSID------DYIIGSMIIYIDIV----YLFAHILMVLIA
AAVIGLTIYA-C--KTKSDFTTKGALLFMCVTSLLLFGIM----AG--VY-YQNV----INLLYSLLCC-LLFGAYLIYDTQLIL-GGSTHKLSID------DYIIGSMIIYIDIV----YLFAHILMVLIA
AAVVGLTFYA-C--KTKSDFTTKGALLFMCTTSLLLFGIM----AG--IY-YQNV----INLLYSLICS-LLFGAYLIYDTQLIL-GGSTHKLSID------DYIIGSMIIYIDIV----YLFAHILMVLIA
LMTMGLTLYA-C--TTKEDFTICTGLLWSLAICLIMLFIF----SL--IY-PSRL----LSIIYSIFAI-FLYSIYIIVDTQLIV-GSKRHSLQKD------DYIIGALILYIDII----ILFLELVKLIAQ
AITIMLTIYA-W--RTKTDYSAAGHFCFVLSMSVLIMCII----GL--FV-RNIW----FHLFICTLCI-IIYGGYIIFDTQLII-GNHSNYLTID------DYIIAAMLLYVDIV----ILFLRILEILMI
AVTASLTVYA-C--RTKSDFTMAGGALFILSSIMFILFIF----AI--FF-FNII----LWLLLCSLSV-ILYGFYLIYDTQLII-GGKSHQLSID------DYVIGTMFIYIDII----ILFLRILQILMI
AITISLTLYA-F--TTKSDITMAGGSLFIFSAVLLVLGLL----CL--IF-NSKI----IHMIYIGGLA-ILYGFYLIYDTQLLM-GNKEYSYSID------DYIVAALQLYIDII----MLFLQLLQLLLE
SLMLSQFLYT-L--TVRYELTYQGTTLFVFGAQILNFHIF----TL--FT-NLSF----YQMVIISFFG-FIFAFYLIFDTQSRV-AGPDYDFNKE------EWRSGTVLVYMDVF----LLILRIGDLLRT
IIVFALSIYA-C--FSKTDVTMKTSLLIYFPLAVIVILIV----AG--SY-QSYM----SQVIVSLAII-GLFSLYLVFDLQRLS-GKKSITYTMD------DYIIAALDIYIDIV----IMFKELIYLLSR
SIVLSLTFLA-H--ATSIDFQFKGTVCSVLGAALAIISVL--------ICIGFPF----IYITYSLLSG-ITFGFYLVFDLHAIM-DGKYEDISLD------DYIIASMLIYVDII----MLFLRTLEVLSR
AIVLFITIYA-C--TTKNDITQKVTAIFYVSMALLVIILV----AS--IF-RSYI----IQTLIGLAIV-GVFCFYLVFDIQRLQ-GNKYLSYSYD------DYIIASLDIYIDIV----VIFQTVLGLANR
GATILLFLYA-C--TTKKDVTIMNSSLFMFISSLLLVSIM----NF--FF-RSEL----LVMLIQYATV-LIYSFYLIYDIQIIM-GDKTLKLDID------NYILGSLIIYIDII----KIFLKVLQLLGQ
GIVVALTIYA-F--KTKTDFTILGGFLFCFVIILIIFGIF----LV--FT-YSRV----AYIVYSALGC-LLYSLYLIYDTQLII-GEKKYSLDID------DYVIGALMLYNNII----YIFFEILRIFRV
GVVVALTIYA-F--KTKTDFTLLGGFLFCFVMVLIIFGIF----LA--FA-YSRT----AYIVYCALGC-LLYSLYLIYDTQLIV-GKKRYALEID------DYVIGALMLYIDII----GLFLEILRLLSS
AMFIALTVYA-F--TTKTDFTVMGGLLFVCLFVFSLAGLF----LL--FT-NNNV----AHIIYCCFGV-IIFSIYIIYDTQLLM-DNKTYSYEID------DYIIASLQLYLDII----NIFLYILEILGR
AIFLSLTLYA-C--TTKSDFTTMGGTLYVIGMGLFIFGFF----LI--FT-NNNV----MHLIYATACA-VLFGFYILYDTQLII-GNKSYKYSID------DYIIASLELYMDII----GLFLQLLEILQR
GITISLTVYA-F--TTKQDFSFCGGLLFVMLSSFILSSIL----LV--FY-NNYV----LEIVACSITA-IIYGIYIVYDTQIVV-GGKYFELSID------DYILGALMLYIDII----RLFLRILEIIIR
VVSISVTIFC-F--QTKVDFTSCTGLFCVLGIVLLVTGIVTSIVLY--FQ-YVYW----LHMLYAALGA-ICFTLFLAYDTQLVL-GNRKHTISPE------DYITGALQIYTDII----YIFTFVLQLMGD
LVCLSVTVFS-F--QTKFDFTSCQGVLFVLLMTLFFSGLILAILLP--FQ-YVPW----LHAVYAALGA-GVFTLFLALDTQLLM-GNRRHSLSPE------EYIFGALNIYLDII----YIFTFFLQLFGT
AVCFTVVIFS-M--QTRYDFTSCMGVLLVSMVVLFIFAIL----CI--FI-RNRI----LEIVYASLGA-LLFTCFLAVDTQLLL-GNKQLSLSPE------EYVFAALNLYTDII----NIFLYILTIIGR
TVFFGLTVYT-L--QSKKDFSKFGAGLFALLWILCLSGFL----KF--FF-YSEI----MELVLAAAGA-LLFCGFIIYDTHSLM-----HKLSPE------EYVLAAISLYLDII----NLFLHLLRFLEA
GIVGGLSTVAMC--APSEKFLNMGAPLGVGLGLVFVSSLG----SM--FLPPTTVAGATLYSVAMYGGL-VLFSMFLLYDTQKVI---KRAEVSPMYGVQKYDPINSMLSIYMDTL----NIFMRVATMLAT
MIFTCFTLSALY--ARRRSYLFLGGILMSALSLLLLSSLG----NV--FF-GSIW----LFQANLYVGL-VVMCGFVLFDTQLII---EKAEHGDQ------DYIWHCIDLFLDFI----TVFRKLMMILAM
TM4
TM5
TM6
TM7
Fig. 6. Multiple alignment of transmembrane Bax inhibitor protein 1 family. Identical and similar residues in at least 50% of the species are indicated in
black and grey, respectively. The alignment includes only the transmembrane (TM) helices and loops. The black bars indicate the predicted transmembrane
helices of human TMBIMs (transmembrane Bax inhibitor motifs). Abbreviations for the taxa are: Pt, Paramecium tetraurelia; TETH, Tetrahymena thermophila;
Hs, Homo sapiens.
MATERIALS AND METHODS
Cells and culture conditions
Experiments were carried out on P. primaurelia, stock 90, cultured at 25°C
in lettuce medium (pH 6.9) inoculated with Enterobacter aerogenes
(Sonneborn, 1970). Cells were harvested in the late log phase of growth.
468
Cell movement analysis
Paramecia were transferred from culture medium into an adaptation
solution containing 1 mmol l−1 CaCl2, 4 mmol l−1 KCl, 4 mmol l−1 NaCl
and 1 mmol l−1 Hepes (pH 7.2) and allowed to equilibrate for 30 min.
Then, they were transferred into a recording camera (internal size:
21×14×3 mm) containing the indicated concentrations of drugs in the
adaptation solution. Responses to drugs were determined by acquiring cellswimming behaviour through an infrared video camera that recorded the
swimming of a small sample of protozoa, i.e. one to five individuals at the
same time. Protozoan movement was digitally recorded for 60 s at
4 frames s−1. Records were analysed by means of image-analysis software
(IToolTrack, e-magine IT s.r.l., Genoa, Italy) to reconstruct each individual
path.
CCR induction
Cells were transferred from culture medium into the adaptation solution for
30 min and then to a bath containing the indicated concentrations of drugs
in the test solution (40 mmol l−1 KCl, 1 mmol l−1 CaCl2, 4 mmol l−1 NaCl,
1 mmol l−1 Hepes, pH 7.2). The presence of high KCl in the test solution
depolarizes the cell membrane, triggering ciliary reversal and backward
swimming. Responses to drugs were determined by transferring individual
The Journal of Experimental Biology
the glycine/TCP-BP protein, the CPP-BP, and the phencycline-binding
protein in mammalian brain synaptosomal membranes to express an
NMDA receptor-like complex, with binding properties similar to those
of classic NMDA receptors (Kumar et al., 1998). Indeed, the NMDAlike receptors elicit Ca2+-dependent responses similar to those
observed for classic NMDA receptors (Michaelis, 1998), which can
be prevented by classic NMDA receptor antagonists (Kumar et al.,
1998; Michaelis, 1998).
In conclusion, these results provide evidence that NMDA-like
receptors, functionally resembling those identified in mammals, are
present in the single-celled organism Paramecium and also suggest
that the glutamatergic NMDA or NMDA-like system is an early
evolved, phylogenetically old mechanism.
RESEARCH ARTICLE
The Journal of Experimental Biology (2014) doi:10.1242/jeb.093914
HsGlyBP
RnGlyBP
GgGlyBP
DrGlyBP
TETHGlyBP
PtGLYBPa
PtGlyBPb
--------------------------MPHKIGFVVVSSSGHEDGFSARELMIHAPTVSGWRSPRFCQFPQEIVLQMVERCRIRKLQLLAHQYMISSKIEFYISESLPEYFAPYQAERFRRLGYVSLCDNEKTGCKARELKSVYVD-AVGQFLKLIFHQNH
--------------------------MPHKIGFVVVSSSGHEDGFSARELMIHAPTVSGWRSPKFCQFPQEIVLQMVERCRIRKLQLLAHQYMISSKVEFYISESLPEYLVPYQAERFRRLGYVSLCDNEKTGCKARELKSVYVD-AVGQFLKLIFHQNH
--------------------------MPHKIGFIVVSSSGHEDGFSAKELMVHAPTVNGWRSPRLCQYPQEIVLQLVERCRIRKLQLLAHQYMISSKIEFFISESLPEYFAPYQSERFHRLGYVPLSDNEKTGFKARELKSVYMD-AVGQYLKLIFHKNY
MRTCHSCQTVVMEMNVGNCYIWLRTGMPHKIGFSVISWSGHEGNYNAKKLMVHAPTVSGWRSTRFCPFPQEIILQLAERCRIRKLQLLAHQYFIPSKVEFHVGDMLPESNSAQQAHSLHRLGYVSLSDNEKTGFRARELKSVHVD-AVGSFLKLTFHRNH
--------------------MSKQQQNMQKLAYKIKSCSSEEQTHRVSELLMQSPQSKGWQSSRFCDYPQEIVLQFHSPVRVRQIQFLSHQYKIASRIEIFVYMPDQNTPFINTEFKYKKLGYLSLDSNEKTGFKSRELKSVYVD-SPALYLKLSFHKNH
------------------------MKGVSKLKYRIVYCSGEDQDYPVTELLTQSPQSRGWQAPKYCEYPQEIAIQFVSAARVRQVQFLSHHCKISTKIELYVHMPDKNIPPQYNQIKYKKLGYLSLDSNERGGYQARELKSVYID-TPCLFMKFVFQKCF
---------------------MNQPKNTRFITFTIPYAQSQDPQFPPSNLLEISSTPLGWQSCRFCQYPQELIFQFQSAITVYKMQILSHEKKIPTKIEVFIGRLQGRMDLENASF--KKIGYFTFHSKEQSNWQARELKTVSIDESNCNYLKLLVHRNH
133
133
133
159
139
135
137
HsGlyBP
RnGlyBP
GgGlyBP
DrGlyBP
TETHGlyBP
PtGLYBPa
PtGlyBPb
VNKYNIYNQVALVAINIIGDPADFSDESN-TASREKLIDHYLGH----NSEDPALEGTYARKS----DYISPLDDLAFDMYQDPEVAQIIRKLDERKREAVQKERYDYAKKLKQAIADLQKVGERLGRYEVEKRCAVEKEDYDLAKEKKQQMEQYRAEVY
ANKYNVYNQVALVAINIIGDPADLGDESN-TTCREKLIDHYLGHSPH-NPEDPALDGTFAGRS----DYISPLDDLAFDMYQDPEVAQIIRRLDERKREAAKKERYDHAKKLKQAIADLQKVGERLGRYEVEKRRAVEKEDYDLAKEKKQQMARYRAQVY
VNKYNLYSQVALVAINIIGDPADYSDDSNKYPSREKLIDHYLGS----KSDDPALDGTYLGKP----DSISPLDDLAFDMYQDPEVAQIIRRLDEKKHEAVHQERYDYAKKLKQAIADLQKVGERLGRYEVEKRYAVEKEDYDLAKKKKQQMDAYRLKVY
VNQYNVYNQVALVALNILGDPIDGSDIGT-TLSRDHLIDQFLNS----SQYSSSLDGTYTGLSSYKCESISPLDDLAFDMYQDPEVAHIIRLLDQKKQVMVREERFDSAKELKQAIADLQKVGERLGRYDVEKLSAIEREDYDTAKQKKEQMDAYRLAVY
NNKYNTFNQIGVIALSCFGDDLTNMK--------------------------------QNIEP-----VKEFYNQIQYQTQF DHVTLDRLRTLEMAKERAVKNDDFDEAQRLKEAIDKLKAIGIQLRQLNERKQIATENEDYDAAKIIKAEIDRLRNAVA
VNKFNLFNQIGVIALSVFGEPLDSPPG-------------------------------YGQMK-----QKEFYNEIQFETQFDQNTLERLRLLEEAKDKAVSREDFMEAKRIKEAIERLKQIGVQLRTLEERKAVAIQNEDYDSASIIKQEIEKLRNAVA
ENKFNPFNQVGIVAIRIYGEKAELPPPKK---------------------------ANKVQDK-----VLDELYNPQQDKLIDTKLLAHIIALEHSKDYAIKQENYQEAKKLKNRITQLRSLGVQLRDLEERKKEVLQNEDYDQAEAIKEQIHKLKIENG
284
287
285
314
262
259
265
HsGlyBP
RnGlyBP
GgGlyBP
DrGlyBP
TETHGlyBP
PtGLYBPa
PtGlyBPb
EQLELHSLLD------AEL-MRRPFDLPLQPLARSGSPCHQKPMPS---------------------------------LPQLEERGTENQF AEPFLQEKPS-SYSLTISPQHSAVDPLLPATDP----HPKINAESLPYDERPLPAIRKHYGEAV---V
EQLELHGLLQ------GEPEMQRPFALPLQPLASPSSPQHWKAVSS---------------------------------LPRTEELAAEDTC AGPILQEKPL-ASS----PRHSAVDRSPPAAGP----APRSHVEALPYDERPLPVTRKQLEEPS---A
QQLQLHNLLD------AELMSRKPPELPVEPVIYDDSLQRTKATNSP-----------------------------SCENTELQTSQGELWK AESLLEEKPADPTSPEPVFRHQSTPPTLSHSTASREVFAQEEAEFLPYDERPLPAIQKHSEEAIRY-L
HQLEIHNLLD------ISQIHRMSGLSDSGFLSPRVGTQKHMPHPP---------------------------------DTHRKKRQG PVKDTDEQDTSKTT---------SPKHTIPSTPFTPP---HVSKIDINSLPYDERPLPTLRNRLSDQSVSEL
PEYLIQKPQPLQYNSNIPYNSNQFQPLMQDPIIPSNQPDMWSSKPVSKPS-----------------NNNFKAENDEYGNQLNKIEEA PLVEQETTEMARQHRSSVQQPQYEESKQDKIQKQKFEEQFMFNNRTNQGSNHDEMQLPAMKNKQNKKPWE-L
PDSMIRRP-----DSAVILN--KLNQQYQQPVYQPQQQYQQQQQQ----------------------QQQYYQQQYQPPPVQSQMAFV PPYQAPPPMMPIQGEEMISQSQFEEQRADPSQMRQKRVAKELN------QHHEDMIVPGALRKQNNNQYP-D
---LVDEQ-----GNEYKPNIDKIRQQELGEQIDNQEDRRNSKPSIK--------------------------QIDNNKIIEDSFDRM PQDEQTYLMEQLNNNNNNITHITNNQNENANTSYQQPVSFDQQLKNDKLLNYDEMVIPALKNKQNNNTQI-L
396
396
409
423
404
383
390
HsGlyBP
RnGlyBP
GgGlyBP
DrGlyBP
TETHGlyBP
PtGLYBPa
PtGlyBPb
EPEMSNADISDARRGGMLGEPEPLTEKALREASSAIDVLGETLVAEAYCKTWSYREDALLALSKKLMEMPVGTPKEDLKNTLRASVFLVRRAIKDIVTSVFQASLKLLKMIITQYIPKHK----LSKLETAHCVERTIPVLLTRTGDSSARLRVTAANFI
EPEVREAD-SDVRRRGVSAEPEPLTEKALREASSAIDTLGEALVAGAYSKMWSCREDALLALYKRLMEMPVGTQKGFVKNMLRASVFLIRRAIKDIVTSVFQASLKLLKMIITQYIPKHK----LGKLDTTYCVERAIPLLLARTGDSSARLRVMALNFI
EPERTEGDISNTPRSGITGEPEPLSEKALREASPAIEVFGEALVSGAYSKSWSYREDALLAVYKKLMEMSVSTPKEDLRNMLRAAIFLVRRAIKDIVSSVFQASLKLLKMIITQYVPKHK----LGKLETSHCVEKTLPGLLSRTGDSSSRLRIVAAKFI
DEILPLADTASPRSPRASGQPEELTEKAQREASLPIEIYGESLVAGAYSKTWSYREDALLAVRKKLMEVPSGSSKAELRSMTRAAVFLCKKALTDKVSSVFLASLNLLKTILSEFIPNHQ----LGKSEISHCVEQTWNNLISRAGDSTSRLRTPAITFI
DDAYANGQAPESVQQNSVGETEQLSGAAKQQAEPLIPVIGDECAAKIFSKSWQIREEGLKWLEAEAQN-PRSINGSDPQALFTAVIGVCSATIADKVAQVSQASMNLLQALCNSRSAKPTSVK----GETGSYVDNCIGLLMEQAGHHVPKVREQAEQAL
DDK----------QQQQQYTTEPLTGDSLQKAEPLIPILTEEFCQKIFSKQWGAREDGLKWLEDQIGR-PTQVNSQDPSIFFLSSIASINYTLGDKVAAVSIRSLSVLQSLLAKYPKIKINKS----AEFNEHIDGILQSLMEKLGEQR---KEQAENAF
EEY--------SEVDKSKQQIEELSSENQKQVEAIKPYYGIDFCKNYFSKNWARREEGIRWLIEQFNN-PTQINLSNIDGAFQATLLLIYKGIQDKADKVVYASLQLMQQTLLKLKPNKLND------ESPIILDNIVLILMEKMGDINERHKEECKKIL
552
551
565
579
559
525
535
HsGlyBP
RnGlyBP
GgGlyBP
DrGlyBP
TETHGlyBP
PtGLYBPa
PtGlyBPb
QEMALFKEVK----SLQIIPSYLVQPLKANSSVHLAMSQMGLLARLLKDLGTG-SSGFTIDNVMKFSVSALEHRVYEVRETAVRIILDMYRQHQASILEYLPPDDS-NTRRNILYKTIFEGFAKIDGR-------ATDAEMRARRKAATEEAEKQKKEEI
QEMALFKEVR----SLQLIPSYLVQPLKTNASVHLAMSQVDLLARLLRDLGTE-GSGFTVDNVMKFAVSALEHRVYEVRETAVRIILDMYRQHPALTLEHLPPDDS-TTRRNLLYKAIFEGFAKIDGR-------PTEAEGKTQKRVVTKEAEKQKKEET
QEMALWSEVK----PLQIVPVHLVQLLKPNSPTHLAMSRVELVECLLKEMGTE-NSGFTISNVMKFATGALEHRVYEVRDVALRIIFGMYRKHKAAILEYLPPDDA-SIRKTVLYKTLFDGFTKIDGK-------LSEAEMRAQKKAATEEAERQKKEEI
QEMALFKEVR----ALQMVPVELVRPMQSSVPARQALSRLELIEKLLEQLGTK-DSGFTLDSIMRFLTGGLEHSSASVRELSMRLIQTVYRLHGKPVLNYLPPDDS-STRKNVLYKNLFDSLAKLDGT--------TINTQKSKKGAERDEGEREK-EEI
FALGSVPIIGNQTIVQSLSKGSGLKPKMQ-SSIKHIVARLNILQQFSKRFSIG-KKDVPLAPVMEYAAKQMVHANNEVRTSAINLITEAYNQVGEGQIEPYIANLP-----EQQRDILSEAFSQNGGG------------GGKNSSPQKQEKKTVVTTNI
LQMADHPSVGPAICVQHLIKGFVGKSKLQ-SSTKHIVGRLAMLTELVKRYEIN-NANMPYQPIVDFAVKLQDDKNEPIRTQAILLLVEVYKFSGN-RLKQSLTNVR-----QAQLDVLEDFFNKIDGG------------GDVDDQPQTNQQRAIIQTNI
LTLAETQIMGSAGVINHLVKGITTKPNLQLSSVRHIQARLMLIYNLIQKYKIN-NERVPYNPVMDVALKYLDHSAEQVRTCAIYIITEIYKNQGD-KVRESIKGIR-----PAQQQILEELFYKIDNG------------GNVQTSFEQEKK--------
699
698
712
724
700
665
668
HsGlyBP
RnGlyBP
GgGlyBP
DrGlyBP
TETHGlyBP
PtGLYBPa
PtGlyBPb
KALQGQLAALKEIQAEVQEKESDAVKPKNQDIQGGKAAPAEALGIPDEH-----------------------------------------YLDNLCIFCGERSESFTE-EGLDLHYWKHCLMLTRCDHCKQVVEISSLTEHLLTECDKKDGFGKCYRCSE
KALQGLSAAPRETQAGVQEKENEAVKLKNQDPQGRKAAPPDTPEIPDNH-----------------------------------------YLDNLCIFCGERNESFTE-EGLDLHYWKHCLMLTRCDHCRQVVEISSLTEHLLTECDKRDGFGKCPRCSE
KVLQGQLAALKEIQAEVQAGKEKESDFQKTKDQGYKSPQPAAAEIPDDHSSV------------------------------------ANYLDN LCIFCGERNESFTE-EGLDLHYWKRCPMLTRCEHCKQVVEIASLTEHLLTECDKKDSFGKCQRCSE
RSLQEQLAVLKEIS---EKGKDNAKVPEKKTEKATKSGVVL-------------------------------------------------VQRS LCIFCGERDESFTE-DGLDLHYWKHCPMLQRCLQCRQVVEIASLTEHLLTECERRTDFSQCPLCSE
QHQGARHDDKGPNKKGKQPSANDKSPSQKDSKKQNNSSKMQASDVME-----------------------------------------------V CEFCHVHNPTFADKKDLDIHLWRECPMLLLCPHCAQVVEVSAYNQHLLEECKKAMKFRQCPRCKE
ESQGAKKGNQ--NQTQNQKPQQQQKQQQQQQYDQNNTAK-------------------------------------------------------- CDYCDRVNPSFRDPDQIDKHLWSECPMLVTCSQCGQVIEIAELTNHLLSECDHKRKFKRCPKCKE
----------------PQPKKKQQQNNPDQVFQQITQA--------------------------------------------------------- CEFCGIENKEFIQSQKLDMHLWKECVMLTTCLSCAQVVEVSQLTNHHFEECEFAKNYRQCETCGC
817
816
835
831
813
767
755
HsGlyBP
RnGlyBP
GgGlyBP
DrGlyBP
TETHGlyBP
PtGLYBPa
PtGlyBPb
AVFKEELPRHIKHKDCNPAKPEKLANRCPLCHENFSPG-EEAWKAHLMGPAGCTMNLRKTHILQKAPALQPGKS---------SAVAASGPLGSKAGSKIPTPKGGLSKSS------SRTYAKR-------AVPKEELPRHIKTKECNPAKSEKVANRCPLCHENFAPG-EEAWKVHLMGSAGCTMNLRKTHILCKAPAPQQGKG---------PMASKSGTSAPKVGSKIPTPKGGLSKSS------SRTHTRR-------AFPKDELPKHIKSRTCNAAKPEHVANHCPLCHDNFSPG-EEAWKSHLMGMDGCAMNLRRLSTINKTIPMQPGKTGGHYLKK--ASPSRTKVRPPSIGSKIPTPRGGPNKSS------GKTYSKQ-------ALMRDKLTEHAQSTACNPPSSDENCNHCPLCHENFTSG-EEGWKSHLMGPEGCKHNSRRRAIQPSTYSYAQGKSVNTAGGKTTMVISESKARGLGGGSRIPAPASRMKRRSRNPPDKASTYRLQQA-----AISVTEYNDHVKEKSCLPHKSPSVANRCPLCHQDIEQG-PEGWRKHLV-VQGCPNNERSTG----------------------------------------------------------------------AILLSGYDKHLED---CRGRNDNTTVRCPLCHQDLKLE-KNTWKNHLI-KQGCLNNERTAG----------------------------------------------------------------------AVLESQLAEHQNK----KICNSSPGDTCPLCYKAIGLN-WKIHLAICE-QQERNKQQPAPQEKK--------------------------------------------------------------------
925
924
950
956
872
823
813
Fig. 7. Sequence alignment of glycine-binding protein (GlyBP) from several organisms. Identical and similar residues in at least 50% of the species are
indicated in black and grey, respectively. The alignment includes full-length sequences for Homo sapiens (Hs), Rattus norvegicus (Rn), Gallus gallus (Gg),
Danio rerio (Dr), P. tetraurelia (Pt) and T. thermophila (TETH).
Drugs used
The drugs used were as follows: NMDA (1–200 μmol l−1), glycine
(1–100 μmol l−1), D-serine (1–100 μmol l−1), MK-801 (1 μmol l−1), D-AP5
(10–200 μmol l−1), CGS19755 (10–40 μmol l−1), DCKA (1 μmol l−1), ZnCl2
(100 nmol l−1), ifenprodil (100 nmol l−1 to 10 μmol l−1), MgCl2
(0.5–3.0 mmol l−1), EGTA (1 mmol l−1) and N-methylglucamine
(8 mmol l−1). MK-801, D-AP5, CGS19755, DCKA and ifenprodil were
purchased from Tocris Bioscience (Bristol, UK). NMDA, glycine, D-serine,
N-methylglucamine and all other chemicals (unless otherwise specified)
were obtained from Sigma Chemical Co. (St Louis, MO, USA). Drugs were
dissolved in milli-Q water.
Statistical analysis
Differences between means (±s.e.m.) were determined using Student’s t-test
(GraphPad Prism, GraphPad, San Diego, CA, USA). Statistical tests were
performed on raw data but, to emphasize any changes in the cell responses
to test solutions, backward swimming durations were normalized to control
(40 mmol l−1 KCl) values (taking the control as 100 s).
Project, http://www.ciliate.org]. Similar searches were also performed using
as queries the relative sequences of the mammalian NMDA receptor-like
complex proteins. Multiple sequence alignments were performed using
M-coffee (Edgar, 2004) and/or ClustalW [http://www.ebi.ac.uk/Tools/
clustalw]. All the sequences used in the sequence alignments are described
in supplementary material Tables S1 and S2. The putative membranespanning alpha helices were assigned using TMHMM software
(http://www.cbs.dtu.dk/services/TMHMM).
Acknowledgements
The authors are grateful to Mrs Maura Agate for her excellent secretarial
assistance.
Competing interests
The authors declare no competing financial interests.
Author contributions
L.G., S.F., M.M. and M.F. performed the experiments. S.C. accomplished the
genetical analysis. P.R., A.M.P., C.U. and G.B. analyzed and discussed the
experimental results and wrote the manuscript.
Funding
This research received no specific grant from any funding agency in the public,
commercial, or not-for-profit sectors.
Supplementary material
Sequence analysis
Supplementary material available online at
http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.093914/-/DC1
TBlastN and BlastP searches (Altschul et al., 1997) using protein sequences
of selected vertebrate, invertebrate and plant glutamate receptors were
conducted on the genomic and protein databases of P. tetraurelia
[Paramecium Genomics, http://paramecium.cgm.cnrs-gif.fr/] and of
Tetrahymena thermophila [The TIGR Tetrahymena thermophila Genome
Aistrup, G. L., Szentirmay, M., Kumar, K. N., Babcock, K. K., Schowen, R. L. and
Michaelis, E. K. (1996). Ion channel properties of a protein complex with
characteristics of a glutamate/N-methyl-D-aspartate receptor. FEBS Lett. 394, 141148.
References
469
The Journal of Experimental Biology
cells with a micropipette into the test solution. Cell responses were recorded
at low power magnification (12×), and then the duration of backward
swimming was scored using a stopwatch.
Tests were carried out on 15–20 cells and were repeated on three to five
different occasions over several weeks.
Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. and
Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein
database search programs. Nucleic Acids Res. 25, 3389-3402.
Ascher, P. and Nowak, L. (1986). A patch-clamp study of excitatory amino acid
activated channels. Adv. Exp. Med. Biol. 203, 507-511.
Ault, B., Evans, R. H., Francis, A. A., Oakes, D. J. and Watkins, J. C. (1980).
Selective depression of excitatory amino acid induced depolarizations by
magnesium ions in isolated spinal cord preparations. J. Physiol. 307, 413-428.
Benveniste, M., Clements, J. D., Vyklický, L., Jr and Mayer, M. L. (1990). A kinetic
analysis of the modulation of N-methyl-D-aspartic acid receptors by glycine in mouse
cultured hippocampal neurones. J. Physiol. 428, 333-357.
Bigiani, A., Delay, R. J., Chaudhari, N., Kinnamon, S. C. and Roper, S. D. (1997).
Responses to glutamate in rat taste cells. J. Neurophysiol. 77, 3048-3059.
Chatterton, J. E., Awobuluyi, M., Premkumar, L. S., Takahashi, H., Talantova, M.,
Shin, Y., Cui, J., Tu, S., Sevarino, K. A., Nakanishi, N. et al. (2002). Excitatory
glycine receptors containing the NR3 family of NMDA receptor subunits. Nature 415,
793-798.
Chiang, A. S., Lin, W. Y., Liu, H. P., Pszczolkowski, M. A., Fu, T. F., Chiu, S. L. and
Holbrook, G. L. (2002). Insect NMDA receptors mediate juvenile hormone
biosynthesis. Proc. Natl. Acad. Sci. USA 99, 37-42.
D’Aniello, A., Spinelli, P., De Simone, A., D’Aniello, S., Branno, M., Aniello, F.,
Fisher, G. H., Di Fiore, M. M. and Rastogi, R. K. (2003). Occurrence and
neuroendocrine role of D-aspartic acid and N-methyl-D-aspartic acid in Ciona
intestinalis. FEBS Lett. 552, 193-198.
Dingledine, R., Borges, K., Bowie, D. and Traynelis, S. F. (1999). The glutamate
receptor ion channels. Pharmacol. Rev. 51, 7-61.
Dryl, S. (1974). Behaviour and motor response of Paramecium. In Paramecium: a
Current Survey (ed J. W. Van Wagtendonk), pp. 165-211. Amsterdam: Elsevier
Science.
Eckert, R. (1972). Bioelectric control of ciliary activity. Science 176, 473-481.
Edgar, R. C. (2004). MUSCLE: a multiple sequence alignment method with reduced
time and space complexity. BMC Bioinformatics 5, 113.
Ezzeddine, Y. and Glanzman, D. L. (2003). Prolonged habituation of the gillwithdrawal reflex in Aplysia depends on protein synthesis, protein phosphatase
activity, and postsynaptic glutamate receptors. J. Neurosci. 23, 9585-9594.
Feinstein, N., Parnas, D., Parnas, H., Dudel, J. and Parnas, I. (1998). Functional
and immunocytochemical identification of glutamate autoreceptors of an NMDA type
in crayfish neuromuscular junction. J. Neurophysiol. 80, 2893-2899.
Fillingham, J. S., Chilcoat, N. D., Turkewitz, A. P., Orias, E., Reith, M. and
Pearlman, R. E. (2002). Analysis of expressed sequence tags (ESTs) in the ciliated
protozoan Tetrahymena thermophila. J. Eukaryot. Microbiol. 49, 99-107.
Grey, K. B., Moss, B. L. and Burrell, B. D. (2009). Molecular identification and
expression of the NMDA receptor NR1 subunit in the leech. Invert. Neurosci. 9, 1120.
Hennessey, T. M. and Kung, C. (1984). An anticalmodulin drug, W-7, inhibits the
voltage-dependent calcium current in Paramecium caudatum. J. Exp. Biol. 110, 169181.
Hui, D., Kumar, K. N., Mach, J. R., Srinivasan, A., Pal, R., Bao, X., Agbas, A.,
Höfner, G., Wanner, K. T. and Michaelis, E. K. (2009). A rat brain bicistronic gene
with an internal ribosome entry site codes for a phencyclidine-binding protein with
cytotoxic activity. J. Biol. Chem. 284, 2245-2257.
Jennings, H. S. (1906). Behavior of the Lower Organisms. Bloomington, IN: Indiana
University Press.
Kano, T., Brockie, P. J., Sassa, T., Fujimoto, H., Kawahara, Y., Iino, Y., Mellem, J.
E., Madsen, D. M., Hosono, R. and Maricq, A. V. (2008). Memory in
Caenorhabditis elegans is mediated by NMDA-type ionotropic glutamate receptors.
Curr. Biol. 18, 1010-1015.
Kay, J. C. and Kass-Simon, G. (2009). Glutamatergic transmission in hydra:
NMDA/D-serine affects the electrical activity of the body and tentacles of Hydra
vulgaris (Cnidaria, Hydrozoa). Biol. Bull. 216, 113-125.
Kumar, K. N., Babcock, K. K., Johnson, P. S., Chen, X., Eggeman, K. T. and
Michaelis, E. K. (1994). Purification and pharmacological and immunochemical
characterization of synaptic membrane proteins with ligand-binding properties of Nmethyl-D-aspartate receptors. J. Biol. Chem. 269, 27384-27393.
Kumar, K. N., Johnson, P. S., Chen, X., Pal, R., Ahmad, M., Ragland, T., Bigge, C.
and Michaelis, E. K. (1998). Cloning of a brain N-methyl-D-aspartate- and D,Lepsilon-2-amino-4-propyl-5-phosphono-3-pentanoic acid (CGP 39653)-binding
protein. Biochem. Biophys. Res. Commun. 253, 463-469.
Le Roith, D., Shiloach, J., Roth, J. and Lesniak, M. A. (1980). Evolutionary origins of
vertebrate hormones: substances similar to mammalian insulins are native to
unicellular eukaryotes. Proc. Natl. Acad. Sci. USA 77, 6184-6188.
Lerma, J., Zukin, R. S. and Bennett, M. V. (1990). Glycine decreases desensitization
of N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytes and is
required for NMDA responses. Proc. Natl. Acad. Sci. USA 87, 2354-2358.
Ly, A. M. and Michaelis, E. K. (1991). Solubilization, partial purification, and
reconstitution of glutamate- and N-methyl-D-aspartate-activated cation channels from
brain synaptic membranes. Biochemistry 30, 4307-4316.
MacDermott, A. B., Mayer, M. L., Westbrook, G. L., Smith, S. J. and Barker, J. L.
(1986). NMDA-receptor activation increases cytoplasmic calcium concentration in
cultured spinal cord neurones. Nature 321, 519-522.
Machemer, H. (1988). Electrophysiology. In Paramecium (ed. H.-D. Görtz), pp. 186215. Berlin: Springer-Verlag.
Machemer, H. and Eckert, R. (1973). Electrophysiological control of reversed ciliary
beating in Paramecium. J. Gen. Physiol. 61, 572-587.
470
The Journal of Experimental Biology (2014) doi:10.1242/jeb.093914
Mayer, M. L. and Westbrook, G. L. (1987). Permeation and block of N-methyl-Daspartic acid receptor channels by divalent cations in mouse cultured central
neurones. J. Physiol. 394, 501-527.
Mayer, M. L., Vyklicky, L., Jr and Clements, J. (1989). Regulation of NMDA receptor
desensitization in mouse hippocampal neurons by glycine. Nature 338, 425-427.
Michaelis, E. K. (1998). Molecular biology of glutamate receptors in the central
nervous system and their role in excitotoxicity, oxidative stress and aging. Prog.
Neurobiol. 54, 369-415.
Mori, H. and Mishina, M. (1995). Structure and function of the NMDA receptor
channel. Neuropharmacology 34, 1219-1237.
Mothet, J. P., Parent, A. T., Wolosker, H., Brady, R. O., Jr, Linden, D. J., Ferris, C.
D., Rogawski, M. A. and Snyder, S. H. (2000). D-serine is an endogenous ligand for
the glycine site of the N-methyl-D-aspartate receptor. Proc. Natl. Acad. Sci. USA 97,
4926-4931.
Murphy, G. G. and Glanzman, D. L. (1997). Mediation of classical conditioning in
Aplysia californica by long-term potentiation of sensorimotor synapses. Science 278,
467-471.
Nahum-Levy, R., Lipinski, D., Shavit, S. and Benveniste, M. (2001). Desensitization
of NMDA receptor channels is modulated by glutamate agonists. Biophys. J. 80,
2152-2166.
Naitoh, Y. (1974). Bioelectric basis of behaviour in protozoa. Am. Zool. 14, 883-893.
Naitoh, Y. and Eckert, R. (1973). Sensory mechanisms in Paramecium. II. Ionic basis
of the hyperpolarizing mechanoreceptor potential. J. Exp. Biol. 59, 53-65.
Nakanishi, S., Masu, M., Bessho, Y., Nakajima, Y., Hayashi, Y. and Shigemoto, R.
(1994). Molecular diversity of glutamate receptors and their physiological functions.
EXS 71, 71-80.
Nam, S. W., Van Noort, D., Yang, Y. and Park, S. (2007). A biological sensor platform
using a pneumatic-valve controlled microfluidic device containing Tetrahymena
pyriformis. Lab Chip 7, 638-640.
Nam, S. W., Kim, S. T., Lee, K. M., Kim, S. H., Kou, S., Lim, J., Hwang, H., Joo, M.
K., Jeong, B., Yoo, S. H. et al. (2009). N-Methyl-D-aspartate receptor-mediated
chemotaxis and Ca2+ signaling in Tetrahymena pyriformis. Protist 160, 331-342.
Neyton, J. and Paoletti, P. (2006). Relating NMDA receptor function to receptor
subunit composition: limitations of the pharmacological approach. J. Neurosci. 26,
1331-1333.
Nomura, T., Tazawa, M., Ohtsuki, M., Sumi-Ichinose, C., Hagino, Y., Ota, A.,
Nakashima, A., Mori, K., Sugimoto, T., Ueno, O. et al. (1998). Enzymes related to
catecholamine biosynthesis in Tetrahymena pyriformis. Presence of GTP
cyclohydrolase I. Comp. Biochem. Physiol. 120B, 753-760.
Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. and Prochiantz, A. (1984).
Magnesium gates glutamate-activated channels in mouse central neurones. Nature
307, 462-465.
Oami, K. and Takahashi, M. (2002). Identification of the Ca2+ conductance
responsible for K+-induced backward swimming in Paramecium caudatum. J.
Membr. Biol. 190, 159-165.
Oliet, S. H. and Mothet, J. P. (2009). Regulation of N-methyl-D-aspartate receptors by
astrocytic D-serine. Neuroscience 158, 275-283.
Paoletti, P. and Neyton, J. (2007). NMDA receptor subunits: function and
pharmacology. Curr. Opin. Pharmacol. 7, 39-47.
Paoletti, P., Ascher, P. and Neyton, J. (1997). High-affinity zinc inhibition of NMDA
NR1-NR2A receptors. J. Neurosci. 17, 5711-5725.
Pierobon, P., Minei, R., Porcu, P., Sogliano, C., Tino, A., Marino, G., Biggio, G. and
Concas, A. (2001). Putative glycine receptors in Hydra: a biochemical and
behavioural study. Eur. J. Neurosci. 14, 1659-1666.
Pierobon, P., Sogliano, C., Minei, R., Tino, A., Porcu, P., Marino, G., Tortiglione, C.
and Concas, A. (2004). Putative NMDA receptors in Hydra: a biochemical and
functional study. Eur. J. Neurosci. 20, 2598-2604.
Preston, R. R. and Saimi, Y. (1990). Calcium ions and regulation of motility in
Paramecium. In Ciliary and Flagellary Membranes (ed. R. A. Bloodgood), pp. 173200. New York, NY: Plenum Publishing Corporation.
Preston, R. R. and Usherwood, P. N. R. (1988). L-glutamate-induced membrane
hyperpolarization and behavioural responses in Paramecium tetraurelia. J. Comp.
Physiol. A 164, 75-82.
Ramoino, P., Fronte, P., Beltrame, F., Diaspro, A., Fato, M., Raiteri, L., Stigliani, S.
and Usai, C. (2003). Swimming behavior regulation by GABAB receptors in
Paramecium. Exp. Cell Res. 291, 398-405.
Ramoino, P., Usai, C., Beltrame, F., Fato, M., Gallus, L., Tagliafierro, G., Magrassi,
R. and Diaspro, A. (2005). GABAB receptor intracellular trafficking after
internalization in Paramecium. Microsc. Res. Tech. 68, 290-295.
Ramoino, P., Gallus, L., Beltrame, F., Diaspro, A., Fato, M., Rubini, P., Stigliani, S.,
Bonanno, G. and Usai, C. (2006). Endocytosis of GABAB receptors modulates
membrane excitability in the single-celled organism Paramecium. J. Cell Sci. 119,
2056-2064.
Renaud, F. L., Chiesa, R., De Jesús, J. M., López, A., Miranda, J. and Tomassini,
N. (1991). Hormones and signal transduction in protozoa. Comp. Biochem. Physiol.
100A, 41-45.
Renaud, F. L., Colon, I., Lebron, J., Ortiz, N., Rodriguez, F. and Cadilla, C. (1995).
A novel opioid mechanism seems to modulate phagocytosis in Tetrahymena. J.
Eukaryot. Microbiol. 42, 205-207.
Rose, J. K., Sangha, S., Rai, S., Norman, K. R. and Rankin, C. H. (2005).
Decreased sensory stimulation reduces behavioral responding, retards
development, and alters neuronal connectivity in Caenorhabditis elegans. J.
Neurosci. 25, 7159-7168.
The Journal of Experimental Biology
RESEARCH ARTICLE
Rosenegger, D. and Lukowiak, K. (2010). The participation of NMDA receptors, PKC,
and MAPK in the formation of memory following operant conditioning in Lymnaea.
Mol. Brain 3, 24.
Scappaticci, A. A. and Kass-Simon, G. (2008). NMDA and GABAB receptors are
involved in controlling nematocyst discharge in hydra. Comp. Biochem. Physiol.
150A, 415-422.
Scappaticci, A. A., Jacques, R., Carroll, J. E., Hufnagel, L. A. and Kass-Simon, G.
(2004). Immunocytochemical evidence for an NMDA1 receptor subunit in
dissociated cells of Hydra vulgaris. Cell Tissue Res. 316, 263-270.
Schell, M. J., Brady, R. O., Jr, Molliver, M. E. and Snyder, S. H. (1997). D-serine as a
neuromodulator: regional and developmental localizations in rat brain glia resemble
NMDA receptors. J. Neurosci. 17, 1604-1615.
Si, A., Helliwell, P. and Maleszka, R. (2004). Effects of NMDA receptor antagonists on
olfactory learning and memory in the honeybee (Apis mellifera). Pharmacol.
Biochem. Behav. 77, 191-197.
Sonneborn, T. M. (1970). Methods in Paramecium research. In Methods in Cell
Physiology, Vol. 4 (ed. D. M. Prescott), pp. 241-339. New York, NY: Academic
Press.
Sonneborn, T. M. (1975). The Paramecium aurelia complex of fourteen sibling
species. Trans. Am. Microscop. Soc. 94, 155-178.
Tikhonov, D. B. and Magazanik, L. G. (2009). Origin and molecular evolution of
ionotropic glutamate receptors. Neurosci. Behav. Physiol. 39, 763-773.
Traynelis, S. F., Wollmuth, L. P., McBain, C. J., Menniti, F. S., Vance, K. M., Ogden,
K. K., Hansen, K. B., Yuan, H., Myers, S. J. and Dingledine, R. (2010). Glutamate
receptor ion channels: structure, regulation, and function. Pharmacol. Rev. 62, 405496.
The Journal of Experimental Biology (2014) doi:10.1242/jeb.093914
Ucieklak, A., Pechè, J., Łopatowska, A. and Wyroba, E. (1993). Effect of propranolol
on the duration of the reversal response in Paramecium octaurelia induced by KCl
and BaCl2. Acta Protozool. 32, 27-32.
Ultsch, A., Schuster, C. M., Laube, B., Betz, H. and Schmitt, B. (1993). Glutamate
receptors of Drosophila melanogaster. Primary structure of a putative NMDA
receptor protein expressed in the head of the adult fly. FEBS Lett. 324, 171-177.
Van Houten, J. L., Yang, W. Q. and Bergeron, A. (2000). Chemosensory signal
transduction in paramecium. J. Nutr. 130 Suppl., S946-S949.
Vyklický, L., Jr, Benveniste, M. and Mayer, M. L. (1990). Modulation of N-methyl-Daspartic acid receptor desensitization by glycine in mouse cultured hippocampal
neurones. J. Physiol. 428, 313-331.
Watkins, J. and Collingridge, G. (1994). Phenylglycine derivatives as antagonists of
metabotropic glutamate receptors. Trends Pharmacol. Sci. 15, 333-342.
Williams, K., Russell, S. L., Shen, Y. M. and Molinoff, P. B. (1993). Developmental
switch in the expression of NMDA receptors occurs in vivo and in vitro. Neuron 10,
267-278.
Wu, C. L., Xia, S., Fu, T. F., Wang, H., Chen, Y. H., Leong, D., Chiang, A. S. and
Tully, T. (2007). Specific requirement of NMDA receptors for long-term memory
consolidation in Drosophila ellipsoid body. Nat. Neurosci. 10, 1578-1586.
Wyroba, E. (1989). β-adrenergic stimulation of phagocytosis in the unicellular
eukaryote Paramecium aurelia. Cell Biol. Int. Rep. 13, 667-678.
Xia, S., Miyashita, T., Fu, T. F., Lin, W. Y., Wu, C. L., Pyzocha, L., Lin, I. R., Saitoe,
M., Tully, T. and Chiang, A. S. (2005). NMDA receptors mediate olfactory learning
and memory in Drosophila. Curr. Biol. 15, 603-615.
Zukin, R. S. and Bennett, M. V. L. (1995). Alternatively spliced isoforms of the
NMDARI receptor subunit. Trends Neurosci. 18, 306-313.
The Journal of Experimental Biology
RESEARCH ARTICLE
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